Biomarkers for Cardiodiabetes

ABSTRACT

The invention provides compositions and methods for determining cardiodiabetes status in a subject. The invention also provides compositions and methods for treating a subject experiencing cardiodiabetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims under 35 USC 119(e) the benefit of U.S.Application 61/151,806, filed Feb. 11, 2009; and U.S. Application61/081,645, filed Jul. 17, 2008, all of which are incorporated byreference in their entirety for all purposes.

FIELD OF THE INVENTION

The invention provides compositions and methods for determiningcardiodiabetes status in a subject. The invention also providescompositions and methods for treating a subject experiencingcardiodiabetes.

BACKGROUND OF THE INVENTION

Metabolic syndrome comprises a number of components that have beenassociated with an increased risk of cardiovascular disease. One type ofmetabolic syndrome is referred to as insulin resistance syndrome orsyndrome X, and is a cluster of risk factors that plays a role incardiovascular disease morbidity among overweight and obese patients andthose persons with type 2 diabetes mellitus. (See generally, Deen,American Family Physician, 2004, 69: 2875-2882.) A report from theNational Cholesterol Education Program-Adult Treatment Panel (NCEP-ATPIII) identified cardiodiabetes as an independent risk factor forcardiovascular disease.

Insulin resistance, abdominal obesity, high blood pressure and lipiddisorders (i.e., elevated levels of triglycerides and low levels ofhigh-density lipoprotein (HDL) cholesterol) are all characteristicsindicative of cardiodiabetes. According to the NCEP-ATP III, a subjecthaving any three of the above abnormalities is deemed to be afflictedwith metabolic syndrome. Table 1 provides a summary of two differentdefinitions for classifying a subject as having metabolic syndrome.

TABLE 1 WHO diagnostic criteria (insulin ATP III diagnostic resistanceplus two criteria (three Component of the following) of the following)Abdominal/central Waist to hip ratio: Waist circumference: obesity >0.90(men), >0.85 >102 cm (40 in) in (women), or BMI >30 men, >88 cm (35 in)kg per m² in women Hypertriglyceridemia ≧150 mg per dL ≧150 mg per dL(≧1.7 mmol per L) Low HDL cholesterol <35 mg per dL (<0.9 <40 mg per dLmmol per L) for men, (<1.036 mmol per L) <39 mg per dL (<1.0 for men,<50 mg per mmol per L) for women dL (<1.295 mmol per L) for women Highblood pressure ≧140/90 mm Hg or ≧130/85 mm Hg or documented use ofdocumented use of antihypertensive antihypertensive therapy therapy Highfasting glucose Impaired glucose ≧110 mg per dL tolerance, impaired(≧6.1 mmol per L) fasting glucose, insulin resistance, or diabetesMicroalbuminuria Urinary albumin to creatinine ratio: 30 mg per g, oralbumin excretion rate: 20 mcg per minute (Reproduced from Deen, supra)

SUMMARY OF THE INVENTION

Patients with insulin resistance and β-cell dysfunction withoutelevation of blood glucose are not identified as suffering from diabetesmellitus. These normoglycemic patients, however, experience the sameelevated cardiovascular risk, which is predominantly linked to vascularinsulin resistance. This condition is newly referred to as“cardiodiabetes” or “cardiocardiodiabetes.” The term “metabolicsyndrome” may also be used herein to refer to this condition. Acardiodiabetic subject might not exhibit one or more of the normalsymptoms of diabetes including, but not limited to, hyperglycemia,fatigue, unexplained weight loss, excessive thirst, excessive urination,excessive eating, poor wound healing, infections, altered mental statusand blurry vision. A cardiodiabetic subject is at high risk forcardiovascular disease and may experience events such as myocardialinfarction and stroke. That is, diabetes mellitus, cardiodiabetes andmetabolic syndrome are phenotypes of a common underlyingpathophysiology.

The cardiodiabetic patient population has experienced a worse outcome inprevious epidemiological studies than those patients who are identifiedand treated as being diabetic. Classical laboratory biomarkers fordiabetes cannot identify these cardiodiabetic patients. Because ofpathophysiological considerations, surrogate markers for glucosemetabolism may not be sufficient to describe the cardiovascular risk ofpatients with cardiodiabetes, and are not sufficient to describe thisrisk for cardiodiabetic patients.

The present provides compositions and methods for detecting and treatinga disease, particularly cardiodiabetes, in a subject. A large number ofbiomarkers are known for a variety of metabolic, diabetic andcardiovascular conditions. See Publication US/2008/0057590, incorporatedby reference in its entirety. However, it has been found thatadiponectin, hsCRP, and intact proinsulin (and optionally leptin orother biomarkers, as outlined below) in combination are useful asbiomarkers for cardiodiabetes, partly because, as discussed below, eachallows the assessment of a different aspect of disease. Each of thesebiomarkers alone does not lead to an overall understanding of asubject's risk of experiencing cardiodiabetes. For example, suppressedadiponectin levels by themselves could indicate a disease state, but notnecessarily. Determining that a subject's adiponectin level issuppressed and hsCRP level is elevated may to some extent suggestcardiodiabetes, but not with the greatest certainty. Further determiningthat a subject's level of intact proinsulin is elevated allows thepractitioner to conclude that the subject has a 200% elevated risk ofcardiovascular disease. Thus, measuring adiponectin, hsCRP and intactproinsulin provides the maximum amount of information concerning thedisease state of a subject through a minimum number of biomarkers.Adiponectin, hsCRP and intact proinsulin also have the practicaladvantage of being physically stable markers. This allows samples to becollected and measured later, for example, in batches, or alternativelythat no special handling of the samples (such as immediate freezing, forexample) need be used.

Accordingly, the present invention provides compositions and methods forthe detection and/or quantification of a set of particular biomarkers(including, but not limited to, any combination of adiponectin, hsCRPand intact proinsulin, as well as combinations including leptin and/orother markers discussed below) that allow for determining cardiodiabetesin a subject. Without such determination, treatment, and thus reductionof serious cardiovascular events, might not otherwise occur.

The invention also provides for selection of efficient risk-reducingtreatment and therapies to avoid cardiovascular complications. Theinvention provides biological markers that in various combinations canbe used in methods to monitor subjects that are undergoing therapiesaffecting cardiodiabetes. Indications of cardiodiabetes allow acaregiver to select or modify therapies or interventions for treatingsubjects. A number of drugs for the treatment of diabetes have beendeveloped and are available on the market. The biomarkers disclosedherein allow for determining a subject's level of response to drugs suchas antidiabetes drugs or other drugs described herein, and formonitoring the effectiveness of drug treatment. The present invention isparticularly directed to the use of a minimum number of biomarkers toprovide a maximum amount of information concerning the disease status ofa subject.

A panel of biomarkers consisting of adiponectin, hsCRP, intactproinsulin (and optionally leptin or other biomarkers, as outlinedbelow) may optionally be combined with measurements of other biomarkersto assess cardiodiabetes.

In current practice, the standard of care is to administer drugs thatnonspecifically reduce symptoms. As these interventions fail in laterstages, more intensive approaches are pursued leading to use of insulinand multiple pharmacotherapy; by then, attempting to treat theunderlying pathophysiology may be ineffective. Thus, the invention alsoprovides a number of drugs and drug combinations that may beadministered to a subject to treat a disease. Through the compositionsand methods of the present invention, subjects may be treated with drugsor drug combinations that not only provide targeted symptom improvement,but also improve a subject's overall disease state.

The invention further provides methods for determining the unsuitabilityof certain drug therapies. That is, depending on measurements of abiomarker panel, a number of drugs and drug combinations that are not orshould not be administered to a subject to treat cardiodiabetes. Theliterature in some instances discloses the administration of certaindrugs to a subject whose biomarker levels satisfy certain criteria asdisclosed herein, whereas according to the present invention, thesesubjects are not or should not be administered these drugs.

Often, subjects may exhibit normal glucose levels but nonethelessexperience β-cell dysfunction, insulin resistance, systemic inflammationand lipostatis. The invention thus provides for early detection ofsubjects with normoglycemic vascular insulin resistance at high risk formyocardial infarction and stroke.

In one aspect, the invention provides a composition comprising a solidsupport comprising: (a) a capture binding ligand selective foradiponectin, (b) a capture binding ligand selective for hsCRP, and (c) acapture binding ligand selective for intact proinsulin.

In one embodiment, one of the capture binding ligands comprises anantibody.

In one embodiment, the composition further comprises: (a) a solublecapture ligand selective for adiponectin, (b) a soluble capture ligandselective for hsCRP, and (c) a soluble capture ligand selective forintact proinsulin.

In one embodiment, each of the soluble capture ligands comprises adetectable marker.

In one embodiment, a detectable marker is a fluorophore.

In one embodiment, a detectable marker is a conjugated enzyme.

In one embodiment, the conjugated enzyme is horseradish peroxidase.

In one embodiment, the composition further comprises a detector.

In one aspect, the invention provides a method of treating acardiodiabetes in a subject comprising (a) measuring the concentrationof a biomarker panel in a sample from the subject, the biomarker panelconsisting of adiponectin, hsCRP and intact proinsulin; and (b)effecting a therapy with respect to the subject.

In one embodiment, if the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, high and high; (b) high, medium and high; (c)medium, high and high; (d) low, high and high; (e) low, medium and high;and (f) low, low and high, then the subject is administered a glitazoneand a drug or combination of drugs selected from a GLP-1 analog and aninsulin.

In one embodiment, if the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, low and high; (b) medium, medium and high;and (c) medium, low and high, then the subject is administered aglitazone and a drug or combination of drugs selected from a GLP-1analog, an insulin and a DPPIV inhibitor.

In one embodiment, if the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, high and low; (b) medium, high and low; (c)low, high and low; (d) low, medium and low; and (e) low, low and low,then the subject is administered metformin and a drug or combination ofdrugs selected from a glitazone, a GLP-1 analog, a DPPIV inhibitor andan insulin.

In one embodiment, if the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, medium and low; (b) high, low and low; (c)medium, medium and low; and (d) medium, low and low, then the therapycomprises administering metformin and a drug or combination of drugsselected from a glitazone, a DPPIV inhibitor and an insulin.

In one embodiment, the subject is not administered a drug or combinationof drugs selected from a sulfonylurea and a glinide.

In one embodiment, the subject is administered one or more additionaldrugs comprising one or more glucose lowering drugs.

In one embodiment, a sample comprises blood.

In one embodiment, the method further comprises taking a measurement ofat least one additional biomarker.

In one embodiment, the additional biomarker is selected from the groupconsisting of leptin, mRNAx, NFκB, IL-6, MMP-9, TNFα, NFκB, eNOS, PPARγ,MCP-1, PAI-1, ICAM/VCAM, E-selectin, P-selectin, von Willebrand factor,sCD40L, insulin, glucose, HbAlc, free fatty acids, triglycerides, VLDL,small dense LDL, oxidized LDL, resistin, HDL, NO, IκB-α, IκB-β, p105,Re1A, TNFα, MIF, inflammatory cytokines and molecules involved insignaling pathways.

In one embodiment, the additional biomarker is MMP-9.

In one embodiment, the concentration of intact proinsulin, hsCRP andadiponectin in the sample is measured and each is a high riskconcentration.

In one embodiment, the additional biomarker is leptin.

In one embodiment, the method comprises contacting the sample with acomposition of invention.

In one aspect, the invention provides use of a composition of theinvention to determine a therapy for a subject experiencingcardiodiabetes.

In one embodiment, the use comprises contacting the composition with asample from the subject and measuring the concentrations of adiponectin,hsCRP and intact proinsulin.

In one embodiment, if the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, high and high; (b) high, medium and high; (c)medium, high and high; (d) low, high and high; (e) low, medium and high;and (f) low, low and high, then the therapy comprises administering aglitazone and a drug or combination of drugs selected from a GLP-1analog and an insulin.

In one embodiment, if the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, low and high; (b) medium, medium and high;and (c) medium, low and high, then the therapy comprises administeringglitazone and a drug or combination of drugs selected from a GLP-1analog, an insulin and a DPPIV inhibitor.

In one embodiment, if the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, high and low; (b) medium, high and low; (c)low, high and low; (d) low, medium and low; and (e) low, low and low,then the therapy comprises administering metformin and a drug orcombination of drugs selected from a glitazone, a GLP-1 analog, an DPPIVinhibitor and an insulin.

In one embodiment, if the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, medium and low; (b) high, low and low; (c)medium, medium and low; and (d) medium, low and low, then the therapycomprises administering metformin and a drug or combination of drugsselected from a glitazone, a DPPIV inhibitor and an insulin.

In one embodiment, the therapy comprises not administering a drug orcombination of drugs selected from a sulfonylurea and a glinide.

In one embodiment, the therapy further comprises administering one ormore additional glucose lowering drugs.

In one aspect, the invention provides a method of determining whether asubject belongs to a population that would benefit from a therapy, themethod comprising contacting a sample from the subject with acomposition of the invention; and measuring the concentrations ofadiponectin, hsCRP and intact proinsulin in the sample.

In one embodiment, the therapy comprises administering a glitazone and adrug or combination of drugs selected from a GLP-1 analog and aninsulin, and wherein if the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, high and high; (b) high, medium and high; (c)medium, high and high; (d) low, high and high; (e) low, medium and high;and (f) low, low and high.

In one embodiment, the therapy comprises administering a glitazone and adrug or combination of drugs selected from a GLP-1 analog, an insulinand a DPPIV inhibitor, and wherein the risk level associated with eachof the concentrations of adiponectin, hsCRP and intact proinsulinrespectively is selected from (a) high, low and high; (b) medium, mediumand high; and (c) medium, low and high.

In one embodiment, the therapy comprises administering metformin and adrug or combination of drugs selected from a glitazone, a GLP-1 analog,a DPPIV inhibitor and an insulin, and wherein the risk level associatedwith each of the concentrations of adiponectin, hsCRP and intactproinsulin respectively is selected from (a) high, high and low; (b)medium, high and low; (c) low, high and low; (d) low, medium and low;and (e) low, low and low.

In one embodiment, the therapy comprises administering metformin and adrug or combination of drugs selected from a glitazone, a DPPIVinhibitor and an insulin, and wherein (a) high, medium and low; (b)high, low and low; (c) medium, medium and low; and (d) medium, low andlow.

In one embodiment, the therapy does not comprise administering a drug orcombination of drugs selected from a sulfonylurea and a glinide.

In one embodiment, the therapy further comprises administering one ormore additional glucose lowering drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a therapy decision matrix for a biomarker panel thatincludes adiponectin, hsCRP and intact proinsulin. The symbol “&” means“and” and “I” means “or.” Specific drugs are provided as non-limitingexamples.

DESCRIPTION OF EMBODIMENTS

Biomarkers

Biomarkers may originate from epidemiological studies, animal studies,pathophysiological considerations and end-organ experiments. Ideally, abiomarker will have a high predictive value for a meaningful outcomemeasure, can be or is validated in appropriately designed prospectivetrials, reflects therapeutic success by corresponding changes in thesurrogate marker results, and should be easy to assess in clinicalpractice.

The term “surrogate marker,” “biomolecular marker,” “biomarker” or“marker” (also sometimes referred to herein as a “target analyte,”“target species” or “target sequence”) refers to a molecule whosemeasurement provides information as to the state of a subject. Invarious exemplary embodiments, the biomarker is used to assess apathological state. Measurements of the biomarker may be used alone orcombined with other data obtained regarding a subject in order todetermine the state of the subject. In one embodiment, the biomarker is“differentially present” in a sample taken from a subject of onephenotypic status (e.g., having a disease) as compared with anotherphenotypic status (e.g., not having the disease). In one embodiment, thebiomarker is “differentially present” in a sample taken from a subjectundergoing no therapy or one type of therapy as compared with anothertype of therapy. Alternatively, the biomarker may be “differentiallypresent” even if there is no phenotypic difference, e.g. the biomarkersmay allow the detection of asymptomatic risk. A biomarker may bedetermined to be “differentially present” in a variety of ways, forexample, between different phenotypic statuses if the mean or medianlevel (particularly the expression level of the associated mRNAs asdescribed below) of the biomarker in the different groups is calculatedto be statistically significant. Common tests for statisticalsignificance include, among others, t-test, ANOVA, Kruskal-Wallis,Wilcoxon, Mann-Whitney and odds ratio.

As described herein, a biomarker may be, for example, a small molecule,an analyte or target analyte, a lipid (including glycolipids), acarbohydrate, a nucleic acid, a protein, any derivative thereof or anyand all combinations of these molecules, with proteins and nucleic acidsfinding particular use in the invention. As will be appreciated by thosein the art, a large number of analytes may be detected using the presentmethods; basically, any biomarker for which a binding ligand, describedbelow, may be made may be detected using the methods of the invention.

In various embodiments, the biomarkers used in the panels of theinvention can be detected either as proteins or as nucleic acids (e.g.mRNA or cDNA transcripts) in any combination. In various embodiments,the protein form of a biomarker is measured. As will be appreciated bythose in the art, protein assays may be done using standard techniquessuch as ELISA assays. In various embodiments, the nucleic acid form of abiomarker (e.g., the corresponding mRNA) is measured. In variousexemplary embodiments, one or more biomarkers from a particular panelare measured using a protein assay and one or more biomarkers from thesame panel are measured using a nucleic acid assay.

As will be appreciated by those in the art, there are a large number ofpossible proteinaceous target analytes and target species that may bedetected using the present invention. The term “protein,” “polypeptide”or “oligopeptide” refers to at least two or more peptides or amino acidsjoined by one or more peptide bonds. The term “protein” includes“peptide.” A protein or an amino acid may be naturally or nonnaturallyoccurring and may be also be an analog, a derivative or a peptidomimeticstructure. The term “protein” refers to wild-type sequences, variants ofwild-type sequences and either of these containing analogs orderivatized amino acids. Examples of derivatized amino acids include,without limitation, those that have been modified by the attachment oflabels (described below); acetylation; acylation; ADP-ribosylation;amidation; covalent attachment of flavin, a heme moiety, a nucleotide, alipid or phosphatidylinositol; cross-linking; cyclization; disulfidebond formation; demethylation; esterification; formation of covalentcrosslinks, cystine or pyroglutamate; formylation; gamma carboxylation;glycosylation; GPI anchor formation; hydroxylation; iodination;methylation; myristoylation; oxidation; proteolytic processing;phosphorylation; prenylation; racemization; selenoylation; sulfation;and ubiquitination. Such modifications are well-known to those of skillin the art and have been described in great detail in the scientificliterature. Several particularly common modifications such asglycosylation, lipid attachment, sulfation, gamma-carboxylation,hydroxylation and ADP-ribosylation, for instance, are described in basictexts, such as Creighton, Proteins—Structure and Molecular Properties,2d ed. (New York: W. H. Freeman and Company, 1993). Many detailedreviews are available on this subject, such as in Johnson, ed.,Posttranslational Covalent Modification of Proteins (New York: AcademicPress, 1983); Seifter et al., Meth. Enzymol., 1990, 182: 626-646; andRattan et al., Ann. N.Y. Acad. Sci., 1992, 663: 48-62. A variant maycontain one or more additions, deletions or substitutions of one or morepeptides compared to wild-type or a different variant sequence. Thesidechains of a protein may be in either the (R) or the (S)configuration. In a preferred embodiment, the amino acids are in the (S)or (L)-configuration. As discussed below, when the protein is used as abinding ligand, it may be desirable to utilize protein analogs to retarddegradation by sample contaminants.

In various embodiments, variants of the sequences described herein,including proteins and nucleic acids based on e.g. splice variants,variants comprising a deletion, addition, substitution, fragments,preproprotein, processed preproprotein (e.g. without a signalingpeptide), processed proprotein (e.g. resulting in an active form),nonhuman sequences and variant nonhuman sequences may be used asbiomarkers.

In various exemplary embodiments, the biomarker is a nucleic acid. Theterm “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein means at least two nucleotides covalently linked together. Anucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, forexample in the use of binding ligand probes, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron, 49(10): 1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35: 3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14: 3487 (1986); Sawai et al, Chem. Lett. 13(5): 805 (1984); Letsingeret al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., ChemicaScripta 26:141 (1986)), phosphorothioate (Mag et al., Nucleic Acids Res.19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briuet al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, (Oxford University Press, 1991), and peptide nucleic acidbackbones and linkages (see Egholm, J. Am. Chem. Soc. 114: 1895 (1992);Meier et al., Chem. Int. Ed. Engl. 31: 1008 (1992); Nielsen, Nature,365: 566 (1993); Carlsson et al., Nature, 380: 207 (1996), all of whichare incorporated by reference). Other analog nucleic acids include thosewith positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995)), non-ionic backbones (U.S. Pat. Nos. 5,386,023; 5,637,684;5,602,240; 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem.Intl. Ed. English 30: 423 (1991); Letsinger et al., J. Am. Chem. Soc.110: 4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13: 1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4: 395 (1994);Jeffs et al., J. Biomolecular NMR 34: 17 (1994); and Horn et al.,Tetrahedron Lett. 37: 743 (1996)) and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al., Chem. Soc. Rev., 24:169-176 (1995)). Several nucleic acid analogs are described in Rawls, C& E News, 35 (June 2, 1997). All of these references are herebyexpressly incorporated by reference. These modifications of theribose-phosphate backbone may be done to increase the stability andhalf-life of such molecules in physiological environments. As will beappreciated by those in the art, all of these nucleic acid analogs mayfind use in the present invention. In addition, mixtures of naturallyoccurring nucleic acids and analogs can be made.

It has been found that assays for cardiodiabetes involving themeasurements of adiponectin, hsCRP and proinsulin has greater value indetermining cardiodiabetes than any of these biomarkers alone. Thisparticular combination of biomarkers allows attainment of clinicallyuseful sensitivity and specificity, and the detection and staging ofless severe cases of a disease that modulates cardiodiabetes.Accordingly, measurements of a biomarker panel comprising or consistingof adiponectin, hsCRP and proinsulin may be used to improve thesensitivity and/or specificity of a diagnostic test compared to a testinvolving any one of these biomarkers alone.

Adiponectin

In various embodiments, adiponectin is used as a biomarker. Adiponectinvalues are useful as a predictive biomarker for insulin resistance andas a monitoring tool in the treatment of insulin resistance relateddisorders. Full-length adiponectin (f-Ad) is a 30 kDa serum proteinspecifically secreted by adipocytes. (See for example, GenBank AccessionNo. BAA08227 incorporated by reference.) Adiponectin typicallycirculates in human blood at concentrations ranging between 5 and 12mg/L, thus accounting for approximately 0.01% of total plasma protein.Schondorf et al., Clin. Lab., 2005, 51: 489-494. Adiponectin levels havehigher median values in females (about 8.7 mg/L) than in males (about5.5 mg/L), and may be affected by age as well. Adiponectin levelscorrelate negatively with BMI, visceral fat mass and insulin levels.Accordingly, adiponectin is decreased in obese subjects and in patientssuffering from type 2 diabetes, macroangiopathy or other metabolicdisorders. The lowest adiponectin values have been found in obesepatients with both type 2 diabetes and coronary heart disease. Tables 2Aand 2B show two different categorization of various adiponectinconcentrations in relation to risk of arteriosclerosis and insulinresistance.

TABLE 2A Adiponectin Concentration Risk Level for Arteriosclerosis(mg/L) and Insulin Resistance >10 low 7-10 medium  <7 high

TABLE 2B Adiponectin Concentration Risk Level for Arteriosclerosis(mg/L) and Insulin Resistance >10 low 7-10 medium 4-7  high  <4 veryhigh

A number of compounds have been shown to affect adiponectin levels in asubject. Pfützner et al., Diabetes, Stoffwechsel and Herz, 2007, 16:91-97 have shown that sulfonylurea, metformin, thiazolidinedione,metformin +sulfonylurea, metformin+thiazolidinedione,sulfonylurea+glitazone, and metformin+sulfonylurea+thiazolidinedione mayhave an effect on adiponectin levels. Thus, in one embodiment, any ofthese compounds or combinations may be administered to a subject.

Accordingly, suitable capture binding ligands, as further discussedherein, for detection and/or quantification of adiponectin include, butare not limited to, antibodies that are selective for adiponectin.Adiponectin antibodies are known and commercially available. In anexemplary embodiment, adiponectin has a peptide sequence according toGenBank Accession No. BAA08227 or is derived from a nucleic acidsequence according to GenBank Accession No. D45371.

High Sensitivity C-Reactive Protein (hsCRP)

In various embodiments, CRP (C-reactive protein) or hsCRP (highsensitivity C-reactive protein) is used as a biomarker. CRP is a memberof the pentraxin family, comprising five noncovalently associatedprotomers arranged symmetrically around a central pore and has amolecular weight of 118,000 Da. (See generally, halal et al.,Hypertension, 2004, 44: 6-11) CRP is a marker of inflammation that hasbeen shown to predict incident myocardial infarction, stroke, peripheralarterial disease, and sudden cardiac death process. Ridker, Circulation,2003, 107: 363-369. Various epidemiological studies involvingindividuals with no prior history of cardiovascular disease have shownthat a single, non-fasting measure of CRP is a strong predictor offuture vascular events. The predictive value of CRP has provenindependent of major traditional risk factors, such as age, smoking,cholesterol levels, blood pressure and diabetes.

Table 3 shows ranges of hsCRP that are correlated with variouscardiovascular risk levels.

TABLE 3 hsCRP Concentration (mg/L) Cardiovascular Risk Level 0-1low >1-3  moderate >3-10 high >10 unspecific elevation; notinterpretable

High-sensitivity CRP assays have been developed and are now widelyavailable. (Roberts et al., Clinical Chemistry 2001, 47: 444-450.) Inone embodiment, a biomarker, such as hsCRP, is measured by immuneturbidometry.

Accordingly, suitable capture binding ligands, as further discussedherein, for detection and/or quantification of hsCRP include, but arenot limited to, antibodies that are selective for hsCRP. hsCRPantibodies are known and commercially available in an exemplaryembodiment, hsCRP has a peptide sequence according to GenBank AccessionNo. AAA52075 or is derived from a nucleic acid sequence according toGenBank Accession No. M11725.

Intact Proinsulin

In various embodiments, intact proinsulin is used as a biomarker. Asused herein, “proinsulin” refers to the prohormone precursor to insulinmade in the β-cell of the islets of Langerhans. Proinsulin may becleaved within β-cell granules to result in two separate molecules:C-peptide and insulin. Partial processing of proinsulin may result insplit or “des” forms of proinsulin. (Clark, Ann Clin Biochem, 1999, 36:541-564.) The term “proinsulin” as used herein preferably refers to theunprocessed form of proinsulin, that is, “intact proinsulin.”

Intact proinsulin concentrations are related to atherosclerosis andcardiovascular disease. If the demand for insulin triggered by insulinresistance reaches a certain threshold, insufficient cleavage capacityof β-cell carboxypeptidase H leads to an increased secretion of intactproinsulin in addition to the desired insulin molecule. Intactproinsulin, however, has been demonstrated to be an independentcardiovascular risk factor. Assessment of β-cell function bydetermination of intact proinsulin facilitates the selection of the mostpromising therapy and also serves to monitor treatment success in thefurther course of the disease. Intact proinsulin may serve as a markerto investigate β-cell function and allows for a secretion-orientedstaging of type 2 diabetes. Table 4 shows the correlation between intactproinsulin concentration and β-cell function.

TABLE 4 Intact Proinsulin Concentration Risk Level of β-cell (pmol/L)Dysfunction >11 high ≧11 low

Table 4 shows that for intact proinsulin concentrations of ≦11 pmol/L,β-cell function can be characterized as good, while for intactproinsulin concentrations of >11 pmol/L, β-cell function can becharacterized as bad.

Chemiluminescence is one technique that can be used to measure intactproinsulin and other biomarkers. Two types of chemiluminescence assaysare able to specifically measure uncleaved “intact” proinsulin and“total” proinsulin (proinsulin and its specific and non-specificdegradation products) in human plasma (MLT Intact Proinsulin and MLTTotal Proinsulin; Sciema, Mainz, Germany). Other methods suitable forproinsulin include without limitation chromatography, particularly HPLC,stable isotope dilution mass spectrometry assays, and ELISA. See,generally, Clark.

Accordingly, suitable capture binding ligands, as further discussedherein, for detection and/or quantification of proinsulin include, butare not limited to, antibodies that are selective for proinsulin. Intactproinsulin antibodies are known and commercially available. In anexemplary embodiment, intact proinsulin has a peptide sequence accordingto GenBank Accession No. AAA72531 or is derived from a nucleic acidsequence according to GenBank Accession No. M12913.

Leptin

In various embodiments, leptin is used as a biomarker. Leptin is a 16kDa adipose-derived protein hormone that plays a role in regulatingenergy intake and energy expenditure, including appetite and metabolism.Leptin, acting through the leptin receptor, is part of a signalingpathway that can inhibit food intake or regulate energy expenditure tomaintain constancy of the adipose mass. Leptin also has severalendocrine functions, and is involved in the regulation of immune andinflammatory responses, hematopoiesis, angiogenesis and wound healing.Mutations in the leptin gene and/or its regulatory regions cause severeobesity, and morbid obesity with hypogonadism. Leptin has also beenlinked to type 2 diabetes mellitus development. Tables 5A and 5B showvarious disease risk levels associated with various concentrations ofleptin in a sample taken from a human subject.

TABLE 5A Leptin Concentration (adult male) (ng/mL) Disease RiskLevel >30 high 20-30 medium <20 low

TABLE 5B Leptin Concentration (adult female) (ng/mL) Disease RiskLevel >60 high 40-60 medium <40 low

Accordingly, suitable capture binding ligands, as further discussedherein, for detection and/or quantification of leptin include, but arenot limited to, antibodies that are selective for leptin. Leptinantibodies are known and commercially available. In an exemplaryembodiment, leptin has a peptide sequence according to RefSeq AccessionNo. NP_(—)000221 or is derived from a nucleic acid sequence according toRefSeq Accession No. NM_(—)000230.

Biomarker Panels

Any combination of the biomarkers described herein is used to assemble abiomarker panel, which is detected or measured as described herein. Asis generally understood in the art, a combination may refer to an entireset or any subset or subcombination thereof. The term “biomarker panel,”“biomarker profile,” or “biomarker fingerprint” refers to a set ofbiomarkers. As used herein, these terms can also refer to any form ofthe biomarker that is measured. Thus, if adiponectin is part of abiomarker panel, then either adiponectin protein or adiponectin mRNA,for example, could be considered to be part of the panel. Whileindividual biomarkers are useful as diagnostics, it has been found thata combination of biomarkers can sometimes provide greater value indetermining a particular status than single biomarkers alone.Specifically, the detection of a plurality of biomarkers in a sample canincrease the sensitivity and/or specificity of the test. Thus, invarious embodiments, a biomarker panel may include 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more types of biomarkers. In various exemplary embodiments,the biomarker panel consists of a minimum number of biomarkers togenerate a maximum amount of information. Thus, in various embodiments,the biomarker panel consists of 3, 4, 5, 6, 7 or 8 types of biomarkers.Where a biomarker panel “consists of a set of biomarkers, no biomarkersother than those of the set are present. In exemplary embodiments, thebiomarker panel consists of 3 biomarkers disclosed herein. In variousembodiments, the biomarker panel consists of 4 biomarkers disclosedherein.

In various exemplary embodiments, the biomarker panel comprisesadiponectin, hsCRP and proinsulin. In various exemplary embodiments, thebiomarker panel comprises any combination of adiponectin, hsCRP andproinsulin. In various exemplary embodiments, the biomarker panelconsists of adiponectin, hsCRP and proinsulin. In various exemplaryembodiments, the biomarker panel consists of any combination ofadiponectin, hsCRP and proinsulin. In various exemplary embodiments, thebiomarker panel consists of 2 biomarkers selected from adiponectin,hsCRP and proinsulin. In various exemplary embodiments, the protein formof adiponectin, hsCRP and proinsulin is detected using a protein assayas known in the art or discussed herein.

In various exemplary embodiments, the biomarker panel comprises orconsists of adiponectin, hsCRP and proinsulin and 1, 2, 3, 4 or moreadditional biomarkers. Such additional biomarkers may, for example,increase the specificity and/or sensitivity the test. Additionalbiomarkers suitable for biomarker panels include, without limitation,any combination of biomarkers selected from leptin, mRNAx, NFκB, IL-6,MMP-9, TNFα, NFκB, eNOS, PPARγ, MCP-1, PAI-1, ICAM/VCAM, E-selectin,P-selectin, von Willebrand factor, sCD40L, insulin, glucose, HbAlc, freefatty acids, triglycerides, VLDL, small dense LDL, oxidized LDL,resistin, HDL, NO, IκB-α, IκB-β, p105, Re1A, TNFα, MIF, inflammatorycytokines, molecules involved in signaling pathways and any biomarkersdisclosed in US Publication US/2008/0057590. It should be understoodthat in this embodiment, the biomarker panel can include any combinationof adiponectin, hsCRP, proinsulin and the remainder of these markers. Invarious exemplary embodiments, the biomarker panel comprisesadiponectin, hsCRP, proinsulin and leptin. In various exemplaryembodiments, the biomarker panel comprises any combination ofadiponectin, hsCRP, proinsulin and leptin. In various exemplaryembodiments, the biomarker panel consists of adiponectin, hsCRP,proinsulin and leptin. In various exemplary embodiments, the biomarkerpanel consists of any combination of adiponectin, hsCRP, proinsulin andleptin. In various exemplary embodiments, the biomarker panel consistsof 2 or 3 biomarkers selected from adiponectin, hsCRP, proinsulin andleptin. In various exemplary embodiments, the biomarker panel comprisesor consists of adiponectin, hsCRP, proinsulin, leptin and 1, 2, 3, 4 ormore additional biomarkers.

The term “clinical parameter” refers to all non-sample or non-analytebiomarkers of subject health status or other characteristics, such as,without limitation, age, ethnicity, gender, diastolic blood pressure andsystolic blood pressure, family history, height, weight, waist and hipcircumference, body-mass index, as well as others such as Type I or TypeII Diabetes Mellitus or Gestational Diabetes Mellitus (collectivelyreferred to here as Diabetes), resting heart rate, homeostatic modelassessment (HOMA), HOMA insulin resistance (HOMA-IR), intravenousglucose tolerance (SI(IVGT)), β-cell function, macrovascular function,microvascular function, atherogenic index, blood pressure, low-densitylipoprotein/high-density lipoprotein ratio, intima-media thickness, andUKPDS risk score. Other clinical parameters are disclosed in USPublication US/2008/0057590.

The biomarkers of the invention show a statistically significantdifference between different states of cardiodiabetes. In variousembodiments, diagnostic tests that use these biomarkers alone or incombination show a sensitivity and specificity of at least about 85%, atleast about 90%, at least about 95%, at least about 98% and about 100%.

Measurement and Detection of Biomarkers

Biomarkers generally can be measured and detected through a variety ofassays, methods and detection systems known to one of skill in the art.The term “measuring,” “detecting,” or “taking a measurement” refers to aquantitative or qualitative determination of a property of an entity,e.g., quantifying the amount or concentration of a molecule or theactivity level of a molecule. The term “concentration” or “level” canrefer to an absolute or relative quantity. Measuring a molecule may alsoinclude determining the absence or presence of the molecule. Variousmethods include but are not limited to refractive index spectroscopy(RI), ultra-violet spectroscopy (UV), fluorescence analysis,radiochemical analysis, near-infrared spectroscopy (near-IR), infrared(IR) spectroscopy, nuclear magnetic resonance spectroscopy (NMR), lightscattering analysis (LS), mass spectrometry, pyrolysis massspectrometry, nephelometry, dispersive Raman spectroscopy, gaschromatography, liquid chromatography, gas chromatography combined withmass spectrometry, liquid chromatography combined with massspectrometry, matrix-assisted laser desorption ionization-time of flight(MALDI-TOF) combined with mass spectrometry, ion spray spectroscopycombined with mass spectrometry, capillary electrophoresis, colorimetryand surface plasmon resonance (such as according to systems provided byBiacore Life Sciences). See also PCT Publications WO/2004/056456 andWO/2004/088309. In this regard, biomarkers can be measured using theabove-mentioned detection methods, or other methods known to the skilledartisan. Other biomarkers can be similarly detected using reagents thatare specifically designed or tailored to detect them.

Different types of biomarkers and their measurements can be combined inthe compositions and methods of the present invention. In variousembodiments, the protein form of the biomarkers is measured. In variousembodiments, the nucleic acid form of the biomarkers is measured. Inexemplary embodiments, the nucleic acid form is mRNA. In variousembodiments, measurements of protein biomarkers are used in conjunctionwith measurements of nucleic acid biomarkers.

Methods for detecting mRNA, such as RT-PCR, real time PCR, branch DNA,NASBA and others, are well known in the art. Using sequence informationprovided by the database entries for the biomarker sequences, expressionof the biomarker sequences can be detected (if present) and measuredusing techniques well known to one of ordinary skill in the art. Forexample, sequences in sequence database entries or sequences disclosedherein can be used to construct probes for detecting biomarker RNAsequences in, e.g., Northern blot hybridization analyses or methodswhich specifically, and, preferably, quantitatively amplify specificnucleic acid sequences. As another example, the sequences can be used toconstruct primers for specifically amplifying the biomarker sequencesin, e.g., amplification-based detection methods such asreverse-transcription based polymerase chain reaction (RT-PCR). Whenalterations in gene expression are associated with gene amplification,deletion, polymorphisms and mutations, sequence comparisons in test andreference populations can be made by comparing relative amounts of theexamined DNA sequences in the test and reference cell populations. Inaddition to Northern blot and RT-PCR, RNA can also be measured using,for example, other target amplification methods (e.g., TMA, SDA, NASBA),signal amplification methods (e.g., bDNA), nuclease protection assays,in situ hybridization and the like.

Thus, of particular interest in the present invention are biochipassays. By “biochip” or “chip” herein is meant a composition generallycomprising a solid support or substrate to which a capture bindingligand (also called an adsorbent, affinity reagent or binding ligand, orwhen nucleic acid is measured, a capture probe) is attached and can bindeither proteins, nucleic acids or both. Generally, where a biochip isused for measurements of protein and nucleic acid biomarkers, theprotein biomarkers are measured on a chip separate from that used tomeasure the nucleic acid biomarkers. For nonlimiting examples ofadditional platforms and methods useful for measuring nucleic acids, seePublications US/2006/0275782, US/2005/0064469 and DE10201463. In variousembodiments, biomarkers are measured on the same platform, such as onone chip. In various embodiments, biomarkers are measured usingdifferent platforms and/or different experimental runs.

By “binding ligand,” “capture binding ligand,” “capture bindingspecies,” “capture probe” or grammatical equivalents herein is meant acompound that is used to detect the presence of or to quantify,relatively or absolutely, a target analyte, target species or targetsequence (all used interchangeably) and that will bind to the targetanalyte, target species or target sequence. Generally, the capturebinding ligand or capture probe allows the attachment of a targetspecies or target sequence to a solid support for the purposes ofdetection as further described herein. Attachment of the target speciesto the capture binding ligand may be direct or indirect. In exemplaryembodiments, the target species is a biomarker. As will be appreciatedby those in the art, the composition of the binding ligand will dependon the composition of the biomarker. Binding ligands for a wide varietyof biomarkers are known or can be readily found using known techniques.For example, when the biomarker is a protein, the binding ligandsinclude proteins (particularly including antibodies or fragments thereof(FAbs, etc.) as discussed further below) or small molecules. The bindingligand may also have cross-reactivity with proteins of other species.Antigen-antibody pairs, receptor-ligands, and carbohydrates and theirbinding partners are also suitable analyte-binding ligand pairs. Invarious embodiments, the binding ligand may be nucleic acid. Nucleicacid binding ligands find particular use when proteins are the targets;alternatively, as is generally described in U.S. Pat. Nos. 5,270,163;5,475,096; 5,567,588; 5,595,877; 5,637,459; 5,683,867; 5,705,337 andrelated patents, hereby incorporated by reference, nucleic acid“aptamers” can be developed for binding to virtually any biomarker.Nucleic acid binding ligands also find particular use when nucleic acidsare binding targets. There is a wide body of literature relating to thedevelopment of binding partners based on combinatorial chemistrymethods. In these embodiments, when the binding ligand is a nucleicacid, preferred compositions and techniques are outlined in PCTPublication WO/1998/020162, hereby incorporated by reference.

In various exemplary embodiments, the capture binding ligand is anantibody. These embodiments are particularly useful for the detection ofthe protein form of a biomarker.

Detecting or measuring the level (e.g. the transcription level) of abiomarker involves binding of the biomarker to a capture binding ligand,generally referred to herein as a “capture probe” when the mRNA of thebiomarker is to be detected on a solid support. In that sense, thebiomarker is a target sequence. The term “target sequence” or “targetnucleic acid” or grammatical equivalents herein means a nucleic acidsequence that may be a portion of a gene, a regulatory sequence, genomicDNA, cDNA, RNA including mRNA and rRNA, or others. As is outlinedherein, the target sequence may be a target sequence from a sample, or asecondary target such as a product of an amplification reaction such asPCR etc. In some embodiments, measuring a nucleic acid can thus refer tomeasuring the complement of the nucleic acid. It may be any length, withthe understanding that longer sequences are more specific.

The target sequence may also comprise different target domains; forexample, a first target domain of the sample target sequence mayhybridize to a first capture probe, a second target domain may hybridizeto a label probe (e.g. a “sandwich assay” format), etc. The targetdomains may be adjacent or separated as indicated. Unless specified, theterms “first” and “second” are not meant to confer an orientation of thesequences with respect to the 5′-3′ orientation of the target sequence.For example, assuming a 5′-3′ orientation of the target sequence, thefirst target domain may be located either 5′ to the second domain, or 3′to the second domain.

When nucleic acids are used as the target analyte, the assays of theinvention can take on a number of embodiments. In one embodiment, theassays are done in solution format, using any number of solution basedformats. In one embodiment, end-point or real time PCR formats are used,as are well known in the art. These assays can be done either as apanel, in individual tubes or wells, or as multiplex assays, using setsof primers and different labels within a single tube or well. Inaddition to PCR-based solution formats, other formats can be utilized,including, but not limited to for example ligation based assaysutilizing FRET dye pairs. In this embodiment, only upon ligation of two(or more) probes hybridized to the target sequence is a signalgenerated.

In many embodiments, the assays are done on a solid support, utilizing acapture probe associated with the surface. As discussed herein, thecapture probes (or capture binding ligands, as they are sometimesreferred to) can be covalently attached to the surface, for exampleusing capture probes terminally modified with functional groups, forexample amino groups, that are attached to modified surfaces such assilanized glass. Alternatively, non-covalent attachment, such aselectrostatic, hydrophobic/hydrophilic adhesion can be utilized. As isappreciated by those in the art and discussed herein, a large number ofattachments are possible on a wide variety of surfaces.

In this embodiment, the assays can take on a number of formats. In oneembodiment, the target sequence comprises a detectable label, asdescribed herein. In this embodiment, the label is generally added tothe target sequence during amplification of the target in one of twoways: either labeled primers are utilized during the amplification stepor labeled dNTPs are used, both of which are well known in the art. Thelabel can either be a primary or secondary label as discussed herein.For example, in one embodiment, the label on the primer and/or a dNTP isa primary label such as a fluorophore. Alternatively, the label may be asecondary label such as biotin or an enzyme; for example, in oneembodiment, the primers or dNTPs are labeled with biotin, and then astreptavidin/label complex is added. In one embodiment, thestreptavidin/label complex contains a label such as a fluorophore. In analternative embodiment, the streptavidin/label complex comprises anenzymatic label. For example, the complex can comprise horseradishperoxidase, and upon addition of TMB, the action of the horseradishperoxidase causes the TMB to precipitate, causing an opticallydetectable event. This has a particular benefit in that the optics fordetection does not require the use of a fluorimeter.

In alternate embodiments, the solid phase assay relies on the use of alabeled soluble capture ligand, sometimes referred to as a “label probe”or “signaling probe” when the target analyte is a nucleic acid. In thisformat, the assay is a “sandwich” type assay, where the capture probebinds to a first domain of the target sequence and the label probe bindsto a second domain. In this embodiment, the label probe can also beeither a primary (e.g. a fluorophore) or a secondary (biotin or enzyme)label. In one embodiment, the label probe comprises biotin, and astreptavidin/enzyme complex is used, as discussed herein. As above, forexample, the complex can comprise horseradish peroxidase, and uponaddition of TMB, the action of the horseradish peroxidase causes the TMBto precipitate, causing an optically detectable event.

Detection of a target species in some embodiments requires a “label” or“detectable marker” (as described below) that can be incorporated in avariety of ways. Thus, in various embodiments, the composition comprisesa “label” or a “detectable marker.” In one embodiment, the targetspecies (or target analyte or target sequence) is labeled; binding ofthe target species thus provides the label at the surface of the solidsupport.

In embodiments finding particular use herein, a sandwich format isutilized, in which target species are unlabeled. In these embodiments, a“capture” or “anchor” binding ligand is attached to the detectionsurface as described herein, and a soluble binding ligand (frequentlyreferred to herein as a “signaling probe,” “label probe” or “solublecapture ligand”) binds independently to the target species and eitherdirectly or indirectly comprises at least one label or detectablemarker.

By “label” or “labeled” herein is meant that a compound has at least onemolecule, element, isotope or chemical compound attached to enable thedetection of the compound. In general, labels fall into four classes: a)isotopic labels, which may be radioactive or heavy isotopes; b)magnetic, electrical, thermal; c) colored or luminescent dyes; and d)enzymes; although labels include particles such as magnetic particles aswell. The dyes may be chromophores or phosphors but are preferablyfluorescent dyes, which due to their strong signals provide a goodsignal-to-noise ratio for decoding. Suitable dyes for use in theinvention include, but are not limited to, fluorescent lanthanidecomplexes, including those of Europium and Terbium, fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,Cascade Blue, Texas Red, Alexa dyes and others described in the 6thEdition of the Molecular Probes Handbook by Richard P. Haugland, herebyexpressly incorporated by reference. Additional labels includenanocrystals or Q-dots as described in U.S. Pat. No. 6,544,732incorporated by reference.

In various embodiments, a secondary detectable label is used. Asecondary label is one that is indirectly detected; for example, asecondary label can bind or react with a primary label for detection,can act on an additional product to generate a primary label (e.g.enzymes), or may allow the separation of the compound comprising thesecondary label from unlabeled materials, etc. Secondary labels include,but are not limited to, one of a binding partner pair; chemicallymodifiable moieties; nuclease inhibitors, enzymes such as horseradishperoxidase, alkaline phosphatases, lucifierases, etc. Secondary labelscan also include additional labels.

In various embodiments, the secondary label is a binding partner pair.For example, the label may be a hapten or antigen, which will bind itsbinding partner. For example, suitable binding partner pairs include,but are not limited to: antigens (such as proteins (including peptides))and antibodies (including fragments thereof (FAbs, etc.)); proteins andsmall molecules, including biotin/streptavidin; enzymes and substratesor inhibitors; other protein-protein interacting pairs;receptor-ligands; and carbohydrates and their binding partners. Nucleicacid-nucleic acid binding proteins pairs are also useful. In general,the smaller of the pair is attached to the NTP for incorporation intothe primer. Preferred binding partner pairs include, but are not limitedto, biotin (or imino-biotin) and streptavidin, digeoxinin and Abs, andProlinx™ reagents.

In the sandwich formats of the invention, an enzyme serves as thesecondary label, bound to the soluble capture ligand. Of particular usein some embodiments is the use of horseradish peroxidase, which whencombined with 3,3′,5,5′-tetramethylbenzidine (TMB) forms a coloredprecipitate which is then detected. In some cases, the soluble captureligand comprises biotin, which is then bound to a enzyme-streptavidincomplex and forms a colored precipitate with the addition of TMB.

In various embodiments, the label or detectable marker is a conjugatedenzyme (for example, horseradish peroxidase). In various embodiments,the system relies on detecting the precipitation of a reaction productor on a change in, for example, electronic properties for detection. Invarious embodiments, none of the compounds comprises a label.

As used herein, the term “fluorescent signal generating moiety” or“fluorophore” refers to a molecule or part of a molecule that absorbsenergy at one wavelength and re-emits energy at another wavelength.Fluorescent properties that can be measured include fluorescenceintensity, fluorescence lifetime, emission spectrum characteristics,energy transfer, and the like.

Signals from single molecules can be generated and detected by a numberof detection systems, including, but not limited to, scanning electronmicroscopy, near field scanning optical microscopy (NSOM), totalinternal reflection fluorescence microscopy (TIRFM), and the like.Abundant guidance is found in the literature for applying suchtechniques for analyzing and detecting nanoscale structures on surfaces,as evidenced by the following references that are incorporated byreference: Reimer et al, editors, Scanning Electron Microscopy: Physicsof Image Formation and Microanalysis, 2nd Edition (Springer, 1998); Nieet al, Anal. Chem., 78: 1528-1534 (2006); Hecht et al, Journal ChemicalPhysics, 112: 7761-7774 (2000); Zhu et al, editors, Near-Field Optics:Principles and Applications (World Scientific Publishing, Singapore,1999); Drmanac, PCT Publication WO/2004/076683; Lehr et al, Anal. Chem.,75: 2414-2420 (2003); Neuschafer et al, Biosensors & Bioelectronics, 18:489-497 (2003); Neuschafer et al, U.S. Pat. No. 6,289,144; and the like.

Thus, a detection system for fluorophores includes any device that canbe used to measure fluorescent properties as discussed above. In variousembodiments, the detection system comprises an excitation source, afluorophore, a wavelength filter to isolate emission photons fromexcitation photons and a detector that registers emission photons andproduces a recordable output, in some embodiments as an electricalsignal or a photographic image. Examples of detection devices includewithout limitation spectrofluorometers and microplate readers,fluorescence microscopes, fluorescence scanners (including e.g.microarray readers) and flow cytometers.

In various exemplary embodiments, the binding of the biomarker to thebinding ligand is specific or selective, and the binding ligand is partof a binding pair. By “specifically bind” or “selectively bind” or“selective for” a biomarker herein is meant that the ligand binds thebiomarker with specificity sufficient to differentiate between thebiomarker and other components or contaminants of the test sample.

The term “solid support” or “substrate” refers to any material that canbe modified to contain discrete individual sites appropriate for theattachment or association of a capture binding ligand. Suitablesubstrates include metal surfaces such as gold, electrodes, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes,Teflon, derivatives thereof, etc.), polysaccharides, nylon ornitrocellulose, resins, mica, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,fiberglass, ceramics, GETEK (a blend of polypropylene oxide andfiberglass) and a variety of other polymers. Of particular use in thepresent invention are the ClonDiag materials described below.

Frequently, the surface of a biochip comprises a plurality ofaddressable locations, each of which comprises a capture binding ligand.An “array location,” “addressable location,” “pad” or “site” hereinmeans a location on the substrate that comprises a covalently attachedcapture binding ligand. An “array” herein means a plurality of capturebinding ligands in a regular, ordered format, such as a matrix. The sizeof the array will depend on the composition and end use of the array.Arrays containing from about two or more different capture bindingligands to many thousands can be made. Generally, the array willcomprise 3, 4, 5, 6, 7 or more types of capture binding ligandsdepending on the end use of the array. In the present invention, thearray can include controls, replicates of the markers and the like.Exemplary ranges are from about 3 to about 50. In some embodiments, thecompositions of the invention may not be in array format; that is, forsome embodiments, compositions comprising a single capture ligand may bemade as well. In addition, in some arrays, multiple substrates may beused, either of different or identical compositions. Thus for example,large arrays may comprise a plurality of smaller substrates.

Accordingly, in one aspect, the invention provides a compositioncomprising a solid support comprising a capture binding ligand for eachbiomarker of a biomarker panel. In various embodiments, the capturebinding ligand is an antibody. In various embodiments, the compositionfurther comprises a soluble binding ligand for each biomarker of abiomarker panel.

A number of different biochip array platforms as known in the art may beused. For example, the compositions and methods of the present inventioncan be implemented with array platforms such as GeneChip (Affymetrix),CodeLink Bioarray (Amersham), Expression Array System (AppliedBiosystems), SurePrint microarrays (Agilent), Sentrix LD BeadChip orSentrix Array Matrix (Illumina) and Verigene (Nanosphere).

In various exemplary embodiments, detection and measurement ofbiomarkers utilizes colorimetric methods and systems in order to providean indication of binding of a target analyte or target species. Incolorimetric methods, the presence of a bound target species such as abiomarker will result in a change in the absorbance or transmission oflight by a sample or substrate at one or more wavelengths. Detection ofthe absorbance or transmission of light at such wavelengths thusprovides an indication of the presence of the target species.

A detection system for colorimetric methods includes any device that canbe used to measure colorimetric properties as discussed above.Generally, the device is a spectrophotometer, a colorimeter or anydevice that measures absorbance or transmission of light at one or morewavelengths. In various embodiments, the detection system comprises alight source; a wavelength filter or monochromator; a sample containersuch as a cuvette or a reaction vial; a detector, such as aphotoresistor, that registers transmitted light; and a display orimaging element.

In various exemplary embodiments, a ClonDiag chip platform is used forthe colorimetric detection of biomarkers. In various embodiments, aClonDiag ArrayTube (AT) is used. One unique feature of the ArrayTube isthe combination of a micro probe array (the biochip) and micro reactionvial. In various embodiments, where a target sequence is a nucleic acid,detection of the target sequence is done by amplifying and biotinylatingthe target sequence contained in a sample and optionally digesting theamplification products. The amplification product is then allowed tohybridize with probes contained on the ClonDiag chip. A solution of astreptavidin-enzyme conjugate, such as Poly horseradish peroxidase (HRP)conjugate solution, is contacted with the ClonDiag chip. After washing,a dye solution such as o-dianisidine substrate solution is contactedwith the chip. Oxidation of the dye results in precipitation that can bedetected colorimetrically. Further description of the ClonDiag platformis found in Monecke S, Slickers P, Hotzel H et al., Clin MicrobiolInfect 2006, 12: 718-728; Monecke S, Berger-Bachi B, Coombs C et al.,Clin Microbiol Infect 2007, 13: 236-249; Monecke S, Leube I and EhrichtR, Genome Lett 2003, 2: 106-118; Monecke S and Ehricht R, Clin MicrobiolInfect 2005, 11: 825-833; German Patent DE 10201463; US PublicationUS/2005/0064469 and ClonDiag, ArrayTube (AT) Experiment Guideline forDNA-Based Applications, version 1.2, 2007, all incorporated by referencein their entirety. One of skill in the art will appreciate that numerousother dyes that react with a peroxidase can be utilized to produce acolorimetric change, such as 3,3′,5,5′-tetramethylbenzidine (TMB). Forinformation on specific assay protocols, seewww.clondiag.com/technologies/publications.php.

In various embodiments, where a target species is a protein, theArrayTube biochip comprises capture binding ligands such as antibodies.A sample is contacted with the biochip, and any target species presentin the sample is allowed to bind to the capture binding ligandantibodies. A soluble capture binding ligand or a detection compoundsuch as a horseradish peroxidase conjugated antibody is allowed to bindto the target species. A dye, such as TMB, is then added and allowed toreact with the horseradish peroxidase, causing precipitation and a colorchange that is detected by a suitable detection device. Furtherdescription of protein detection using ArrayTube is found in, forexample, Huelseweh B, Ehricht R and Marschall H-J, Proteomics, 2006, 6,2972-2981; and ClonDiag, ArrayTube (AT) Experiment Guideline forProtein-Based Applications, version 1.2, 2007, all incorporated byreference in their entirety.

Transmission detection and analysis is performed with a ClonDiag ATreader instrument. Suitable reader instruments and detection devicesinclude the ArrayTube Workstation ATS and the ATR 03.

In addition to ArrayTube, the ClonDiag ArrayStrip (AS) can be used. TheArrayStrip provides a 96-well format for high volume testing. EachArrayStrip consists of a standard 8-well strip with a microarrayintegrated into the bottom of each well. Up to 12 ArrayStrips can beinserted into one microplate frame enabling the parallel multiparametertesting of up to 96 samples. The ArrayStrip can be processed using theArrayStrip Processor ASP, which performs all liquid handling,incubation, and detection steps required in array based analysis. Invarious embodiments, where a protein is detected, a method of using theArrayStrip to detect the protein comprises conditioning the AS arraywith buffer or blocking solution; loading of up to 96 sample solutionsin the AS wells to allow for binding of the protein; 3× washing;conjugating with a secondary antibody linked to HRP; 3× washing;precipitation staining with TMB; and AS array imaging and optional datastorage.

Those skilled in the art will be familiar with numerous additionalimmunoassay formats and variations thereof which may be useful forcarrying out the method disclosed herein. See generally E. Maggio,Enzyme-Immunoassay, (CRC Press, Inc., Boca Raton, Fla., 1980); see alsoU.S. Pat. Nos. 4,727,022; 4,659,678; 4,376,110; 4,275,149; 4,233,402;and 4,230,767.

In general, immunoassays carried out in accordance with the presentinvention may be homogeneous assays or heterogeneous assays. In ahomogeneous assay the immunological reaction usually involves thespecific antibody (e.g., anti-biomarker protein antibody), a labeledanalyte, and the sample of interest. The signal arising from the labelis modified, directly or indirectly, upon the binding of the antibody tothe labeled analyte. Both the immunological reaction and detection ofthe extent thereof can be carried out in a homogeneous solutionImmunochemical labels which may be employed include free radicals,radioisotopes, fluorescent dyes, enzymes, bacteriophages, or coenzymes.

In a heterogeneous assay approach, the reagents are usually the sample,the antibody, and means for producing a detectable signal. Samples asdescribed above may be used. The antibody can be immobilized on asupport, such as a bead (such as protein A and protein G agarose beads),plate or slide, and contacted with the specimen suspected of containingthe antigen in a liquid phase. The support is then separated from theliquid phase and either the support phase or the liquid phase isexamined for a detectable signal employing means for producing suchsignal. The signal is related to the presence of the analyte in thesample. Means for producing a detectable signal include the use ofradioactive labels, fluorescent labels, or enzyme labels. For example,if the antigen to be detected contains a second binding site, anantibody which binds to that site can be conjugated to a detectablegroup and added to the liquid phase reaction solution before theseparation step. The presence of the detectable group on the solidsupport indicates the presence of the antigen in the test sample.Examples of suitable immunoassays include immunoblotting,immunofluorescence methods, immunoprecipitation, chemiluminescencemethods, electrochemiluminescence (ECL) or enzyme-linked immunoassays.

Antibodies can be conjugated to a solid support suitable for adiagnostic assay (e.g., beads such as protein A or protein G agarose,microspheres, plates, slides or wells formed from materials such aslatex or polystyrene) in accordance with known techniques, such aspassive binding. Antibodies as described herein may likewise beconjugated to detectable labels or groups such as radiolabels (e.g.,³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkalinephosphatase), and fluorescent labels (e.g., fluorescein, Alexa, greenfluorescent protein, rhodamine) in accordance with known techniques.

As used herein, the term “antibody” means a protein comprising one ormore polypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (λ) and heavy chain geneticloci, which together compose the myriad variable region genes, and theconstant region genes mu (μ), delta (δ), gamma (↓), epsilon (ε) andalpha (α), which encode the IgM, IgD, IgG, IgE, and IgA isotypesrespectively. Antibody herein is meant to include full length antibodiesand antibody fragments, and may refer to a natural antibody from anyorganism, an engineered antibody or an antibody generated recombinantlyfor experimental, therapeutic or other purposes as further definedbelow. Antibody fragments include Fab, Fab′, F(ab′)₂, Fv, scFv or otherantigen-binding subsequences of antibodies and can include thoseproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA technologies. The term “antibody” refers toboth monoclonal and polyclonal antibodies. Antibodies can beantagonists, agonists, neutralizing, inhibitory or stimulatory.

Using any of the methods and compositions described herein, a sample canbe assayed to determine levels of a biomarker panel. Thus, in oneaspect, the invention provides a method of assaying a sample from apatient to determine concentrations of a biomarker panel in the sample.In some embodiments, the method comprises contacting the sample with acomposition comprising a solid support comprising a capture bindingligand or capture probe for each biomarker of a biomarker panel.

The invention further provides kits for use in determiningcardiodiabetes for a number of medical (including diagnostic andtherapeutic), industrial, forensic and research applications. Kits maycomprise a carrier, such as a box, carton, tube or the like, having inclose confinement therein one or more containers, such as vials, tubes,ampoules, bottles, pouches, envelopes and the like. In variousembodiments, the kits comprise one or more components selected from oneor more media or media ingredients and reagents for the measurement ofthe various biomarkers and biomarker panels disclosed herein. Forexample, kits of the invention may also comprise, in the same ordifferent containers, one or more DNA polymerases, one or more primers,one or more suitable buffers, one or more nucleotides (such asdeoxynucleoside triphosphates (dNTPs) and preferably fluorescentlylabeled dNTPs) and labeling components. The one or more components maybe contained within the same container, or may be in separate containersto be admixed prior to use. The kits of the present invention may alsocomprise one or more instructions or protocols for carrying out themethods of the present invention. The kits may also comprise a computeror a component of a computer, such as a computer-readable storage mediumor device. Examples of storage media include, without limitation,optical disks such as CD, DVD and Blu-ray Discs (BD); magneto-opticaldisks; magnetic media such as magnetic tape and internal hard disks andremovable disks; semi-conductor memory devices such as EPROM, EEPROM andflash memory; and RAM. The computer-readable storage medium may comprisesoftware encoding references to the various therapies and treatmentregimens disclosed herein. The software may be interpreted by a computerto provide the practitioner with treatments according to variousmeasured concentrations of biomarkers as provided herein. In variousembodiments, the kit comprises a biomarker assay involving alateral-flow-based point-of-care rapid test with detection of riskthresholds, or a biochip with quantitative assays for the constituentbiomarkers.

Methods of Diagnosing and Treating

The compositions and methods of the present invention can be used in theprognosis, diagnosis and treatment of disease in a subject. Theinvention provides compositions and methods for laboratory andpoint-of-care tests for measuring biomarkers in a sample from a subject.The invention can be generally applied for a number of differentdiseases. In exemplary embodiments, the disease is cardiodiabetes. Inexemplary embodiments, the disease is cardiovascular disease or risk. Inexemplary embodiments, the disease is metabolic syndrome.

The biomarkers and biomarker panels disclosed herein can be used inmethods to diagnose, identify or screen subjects that have, do not haveor are at risk for having disease; to monitor subjects that areundergoing therapies for disease; to determine or suggest a new therapyor a change in therapy; to differentially diagnose disease statesassociated with the disease from other diseases or withinsub-classifications of disease; to evaluate the severity or changes inseverity of disease in a patient; to stage a subject with the diseaseand to select or modify therapies or interventions for use in treatingsubjects with the disease. In an exemplary embodiment, the methods ofthe present invention are used to identify and/or diagnose subjects whoare asymptomatic or presymptomatic for a disease. In this context,“asymptomatic” or “presymptomatic” means not exhibiting the traditionalsymptoms or enough abnormality for disease. In exemplary embodiments,the subject is normoglycemic.

In various embodiments, a method of determining a prognosis of a diseasein a subject, diagnosing a disease in a subject, or treating a diseasein a subject comprises taking a measurement of a biomarker panel in asample from the subject. In various exemplary embodiments, the biomarkerpanel consists of adiponectin, hsCRP and intact proinsulin.

The term “disease status” includes any distinguishable manifestation ofthe disease, including non-disease. For example, disease statusincludes, without limitation, the presence or absence of disease, therisk of developing disease, the stage of the disease, the progression ofdisease (e.g., progress of disease or remission of disease over time),the severity of disease and the effectiveness or response to treatmentof disease.

A “subject” in the context of the present invention is an animal,preferably a mammal The mammal can be a human, non-human primate, mouse,rat, dog, cat, horse, or cow, but are not limited to these examples. Invarious exemplary embodiments, a subject is human and may be referred toas a patient. Mammals other than humans can be advantageously used assubjects that represent animal models of a disease or for veterinarianapplications. A subject can be one who has been previously diagnosed oridentified as having a disease, and optionally has already undergone, oris undergoing, a therapeutic intervention for a disease. Alternatively,a subject can also be one who has not been previously diagnosed ashaving a disease. For example, a subject can be one who exhibits one ormore risk factors for a disease, or one who does not exhibit a diseaserisk factor, or one who is asymptomatic for a disease. A subject canalso be one who is suffering from or at risk of developing a disease. Incertain embodiments, the subject can be already undergoing therapy orcan be a candidate for therapy.

As will be appreciated by those in the art, the biomarkers may bemeasured in using several techniques designed to achieve morepredictable subject and analytical variability. On subject variability,many of the above biomarkers are commonly measured in a fasting state,commonly in the morning, providing a reduced level of subjectvariability due to both food consumption and metabolism and diurnalvariation. All fasting and temporal-based sampling procedures using thebiomarkers described herein may be useful for performing the invention.Pre-processing adjustments of biomarker results may also be intended toreduce this effect.

The term “sample” refers to a specimen or culture obtained from asubject and includes fluids, gases and solids including for exampletissue. In various exemplary embodiments, the sample comprises blood.Fluids obtained from a subject include for example whole blood or ablood derivative (e.g. serum, plasma, or blood cells), ovarian cystfluid, ascites, lymphatic, cerebrospinal or interstitial fluid, saliva,mucous, sputum, sweat, urine, or any other secretion, excretion, orother bodily fluids. As will be appreciated by those in the art,virtually any experimental manipulation or sample preparation steps mayhave been done on the sample. For example, wash steps and/orfragmentation may be applied to a sample. In various embodiments, abiomarker panel is measured directly in a subject without the need toobtain a separate sample from the patient.

In one aspect, the invention provides a method of diagnosing a subjectfor a disease comprising taking a measurement of a biomarker panel; andcorrelating the measurement with the disease. The term “correlating”generally refers to determining a relationship between one type of datawith another or with a state. In various embodiments, correlating themeasurement with disease comprises comparing the measurement with areference biomarker profile or some other reference value. In variousembodiments, correlating the measurement with disease comprisesdetermining whether the subject is currently in a state of disease.

The quantity or activity measurements of a biomarker panel can becompared to a reference value. Differences in the measurements ofbiomarkers in the subject sample compared to the reference value arethen identified. In exemplary embodiments, the reference value is givenby a risk category as described further below.

In various embodiments, the reference value is a baseline value. Abaseline value is a composite sample of an effective amount ofbiomarkers from one or more subjects who do not have a disease, who areasymptomatic for a disease or who have a certain level of a disease. Abaseline value can also comprise the amounts of biomarkers in a samplederived from a subject who has shown an improvement in risk factors of adisease as a result of treatments or therapies. In these embodiments, tomake comparisons to the subject-derived sample, the amounts ofbiomarkers are similarly calculated. A reference value can also comprisethe amounts of biomarkers derived from subjects who have a diseaseconfirmed by an invasive or non-invasive technique, or are at high riskfor developing a disease. Optionally, subjects identified as having adisease, or being at increased risk of developing a disease are chosento receive a therapeutic regimen to slow the progression of a disease,or decrease or prevent the risk of developing a disease. A disease isconsidered to be progressive (or, alternatively, the treatment does notprevent progression) if the amount of biomarker changes over timerelative to the reference value, whereas a disease is not progressive ifthe amount of biomarkers remains constant over time (relative to thereference population, or “constant” as used herein). The term “constant”as used in the context of the present invention is construed to includechanges over time with respect to the reference value.

The biomarkers of the present invention can be used to generate a“reference biomarker profile” of those subjects who do not have adisease according to a certain threshold, are not at risk of having adisease or would not be expected to develop a disease. The biomarkersdisclosed herein can also be used to generate a “subject biomarkerprofile” taken from subjects who have a disease or are at risk forhaving a disease. The subject biomarker profiles can be compared to areference biomarker profile to diagnose or identify subjects at risk fordeveloping a disease, to monitor the progression of disease, as well asthe rate of progression of disease, and to monitor the effectiveness ofdisease treatment modalities. The reference and subject biomarkerprofiles of the present invention can be contained in a machine-readablemedium, such as but not limited to, analog tapes like those readable bya VCR; optical media such as CD-ROM, DVD-ROM and the like; and solidstate memory, among others.

Measurements of the biomarker panels of the invention can lead apractitioner to effect a therapy with respect to a subject. Thus, theinvention provides methods of treating a disease in a subject comprisingtaking a measurement of a biomarker panel in a sample from the subject,and effecting a therapy with respect to the subject. The terms “therapy”and “treatment” may be used interchangeably. In certain embodiments, thetherapy can be selected from, without limitation, initiating therapy,continuing therapy, modifying therapy or ending therapy. A therapy alsoincludes any prophylactic measures that may be taken to prevent disease.

In certain embodiments, therapy comprises administering adisease-modulating drug to a subject. The drug can be a therapeutic orprophylactic used in subjects diagnosed or identified with a disease orat risk of having the disease. In certain embodiments, modifying therapyrefers to altering the duration, frequency or intensity of therapy, forexample, altering dosage levels.

In various embodiments, effecting a therapy comprises causing a subjectto or communicating to a subject the need to make a change in lifestyle,for example, increasing exercise, changing diet, reducing or eliminatingsmoking and so on. The therapy can also include surgery, for example,bariatric surgery.

Measurement of biomarker levels allow for the course of treatment of adisease to be monitored. The effectiveness of a treatment regimen for adisease can be monitored by detecting one or more biomarkers in aneffective amount from samples obtained from a subject over time andcomparing the amount of biomarkers detected. For example, a first samplecan be obtained prior to the subject receiving treatment and one or moresubsequent samples are taken after or during treatment of the subject.Changes in biomarker levels across the samples may provide an indicationas to the effectiveness of the therapy.

To identify therapeutics or drugs that are appropriate for a specificsubject, a test sample from the subject can also be exposed to atherapeutic agent or a drug, and the level of one or more biomarkers canbe determined Biomarker levels can be compared to a sample derived fromthe subject before and after treatment or exposure to a therapeuticagent or a drug, or can be compared to samples derived from one or moresubjects who have shown improvements relative to a disease as a resultof such treatment or exposure. Thus, in one aspect, the inventionprovides a method of assessing the efficacy of a therapy with respect toa subject comprising taking a first measurement of a biomarker panel ina first sample from the subject; effecting the therapy with respect tothe subject; taking a second measurement of the biomarker panel in asecond sample from the subject and comparing the first and secondmeasurements to assess the efficacy of the therapy.

Additionally, therapeutic or prophylactic agents suitable foradministration to a particular subject can be identified by detecting abiomarker (which may be two or more) in an effective amount from asample obtained from a subject and exposing the subject-derived sampleto a test compound that determines the amount of the biomarker(s) in thesubject-derived sample. Accordingly, treatments or therapeutic regimensfor use in subjects having a disease or subjects at risk for developinga disease can be selected based on the amounts of biomarkers in samplesobtained from the subjects and compared to a reference value. Two ormore treatments or therapeutic regimens can be evaluated in parallel todetermine which treatment or therapeutic regimen would be the mostefficacious for use in a subject to delay onset, or slow progression ofa disease. In various embodiments, a recommendation is made on whetherto initiate or continue treatment of a disease.

Drug Treatments

In various exemplary embodiments, effecting a therapy comprisesadministering a disease-modulating drug to the subject. The subject maybe treated with one or more disease-modulating drugs until alteredlevels of the measured biomarkers return to a baseline value measured ina population not suffering from the disease, experiencing a less severestage or form of a disease or showing improvements in disease biomarkersas a result of treatment with a disease-modulating drug. Additionally,improvements related to a changed level of a biomarker or clinicalparameter may be the result of treatment with a disease-modulating drugand may include, for example, a reduction in body mass index (BMI), areduction in total cholesterol levels, a reduction in LDL levels, anincrease in HDL levels, a reduction in systolic and/or diastolic bloodpressure, or combinations thereof

A number of compounds such as a disease-modulating drug may be used totreat a subject and to monitor progress using the methods of theinvention. In certain embodiments, the disease-modulating drug comprisesan antiobesity drug, a β-blocker, an angiotensin-converting enzyme (ACE)inhibitor, a diuretic, a calcium channel blocker, an angiotensin IIreceptor blocker, an antiplatelet agent, an anti-coagulant agent, asulfonylurea (SU), a biguanide, an insulin, a glitazone(thiazolidinedione (TZD)), a nitrate, an non-steroidal anti-inflammatoryagent, a statin, cilostazol, pentoxifylline, buflomedil ornaftidrofuryl. In addition, any and all combinations of these drugs maybe administered.

The beneficial effects of these and other drugs can be visualized byassessment of clinical and laboratory biomarkers. For example, resultsfrom PROactive (Pfützner et al., Expert Review of CardiovascularTherapy, 2006, 4: 445-459) and recent metanalyses have shown that thesesurrogate changes may translate into effective reduction ofcardiovascular risk in patients with type 2 diabetes mellitus.

In various exemplary embodiments, a glitazone (also referred to as athiazolidinedione (TZD)) is administered to a subject to treat adisease. The glitazones form a class of drugs that have been used totreat subjects with diabetes mellitus (type 2) and related diseases.Glitazones act by binding to PPARs (peroxisome proliferator-activatedreceptors), a group of receptor molecules inside the cell nucleus,specifically PPARγ (gamma) The normal ligands for these receptors arefree fatty acids (FFAs) and eicosanoids. When activated, the receptormigrates to the DNA, activating transcription of a number of specificgenes.

Examples of glitazones that may be useful in the present inventioninclude, but are not limited to, rosiglitazone (Avandia™), pioglitazone(Actos™) and troglitazone (Rezulin™). Glitazones and other drugsadministered to treat a subject have been shown to affect levels ofvarious biomarkers. In various exemplary embodiments, pioglitazone isadministered to a subject.

Furthermore, a glitazone such as pioglitazone may also be administeredwith other drugs. In various embodiments, pioglitazone is administeredwith a statin, including but not limited to simvastatin. In variousembodiments, pioglitazone may be administered with another glitazone,such as rosiglitazone. In various embodiments, pioglitazone may beadministered with an oral antidiabetic drug, including but not limitedto a sulfonylurea (such as glimepiride) or a biguanide (such asmetformin)

In various embodiments, a glucagon-like pepide 1 (GLP-1) analog isadministered to a subject to treat a disease. Examples of GLP-1 analogsinclude but are not limited to exenatide and liraglutide.

In various embodiments, a dipeptidyl peptidase IV (DPPIV) inhibitor isadministered to a subject to treat a disease. Examples of DPPIVinhibitors include but are not limited to sitagliptin, vildagliptin andsaxagliptin.

In various embodiments, metformin is administered to a subject to treata disease.

In various embodiments, a glinide is administered to a subject to treata disease. Examples of glinides include but are not limited torepgalinide and nateglinide.

In various embodiments, a sulfonylurea is administered to a subject totreat a disease. Examples of sulfonylureas include but are not limitedto gliclazide and glimepiride.

In various embodiments, an α-glucosidase inhibitor is administered to asubject to treat a disease. An example of an α-glucosidase inhibitor isacarbose.

In various embodiments, an insulin is administered to a subject to treata disease. The term “insulin” by itself refers to any naturallyoccurring form of insulin as well as any derivatives and analogs thereofDifferent types of insulin may vary in the onset, peak occurrence andduration of their effects. Examples of insulin that may be useful in thepresent invention include but are not limited to regular human insulin,intermediate acting regular human insulin (e.g., NPH human insulin),Zn-retarded insulin, short acting insulin analog and long acting insulinanalog. Examples of Zn-retarded insulin include but are not limited tolente and ultralente. Examples of short-acting insulin analog includebut are not limited to lispro, aspart and glulisine. Examples oflong-acting insulin analog include but are not limited to glargine andlevemir.

Any drug or combination of drugs disclosed herein may be administered toa subject to treat a disease. The drugs herein can be formulated in anynumber of ways, often according to various known formulations in the artor as disclosed or referenced herein.

In various exemplary embodiments, a subject is administered a glitazoneand a drug or combination of drugs selected from a GLP-1 analog and aninsulin. In various exemplary embodiments, a subject is administered aglitazone and a drug or combination of drugs selected from a GLP-1analog, an insulin and a DPPIV inhibitor. In various exemplaryembodiments, a subject is administered metformin and a drug orcombination of drugs selected from a glitazone, a GLP-1 analog, a DPPIVinhibitor and an insulin. In various exemplary embodiments, a subject isadministered metformin and a drug or combination of drugs selected froma glitazone, a DPPIV inhibitor and an insulin.

In various embodiments, a subject is administered a drug or combinationof drugs selected from a glitazone, a GLP-1 analog and an insulin. Invarious embodiments, a subject is administered a drug or combination ofdrugs selected from a glitazone, a GLP-1 analog, an insulin and a DPPIVinhibitor. In various embodiments, a subject is administered a drug orcombination of drugs selected from metformin, a glitazone, a GLP-1analog, a DPPIV inhibitor and an insulin. In various embodiments, asubject is administered a drug or combination of drugs selected frommetformin, a glitazone, a DPPIV inhibitor and an insulin.

In various embodiments, any drug or combination of drugs disclosedherein is not administered to a subject to treat a disease. In theseembodiments, the practitioner may refrain from administering the drug orcombination of drugs, may recommend that the subject not be administeredthe drug or combination of drugs or may prevent the subject from beingadministered the drug or combination of drugs.

In various exemplary embodiments, a glinide is not administered to asubject. In various exemplary embodiments, a sulfonylurea is notadministered to a subject. In various exemplary embodiments, a drug ordrug combination selected from a glinide and a sulfonylurea is notadministered to a subject.

In various embodiments, one or more additional drugs may be optionallyadministered in addition to those that are recommended or have beenadministered. An additional drug will typically not be any drug that isnot recommended or that should be avoided. In exemplary embodiments, oneor more additional drugs comprise one or more glucose lowering drugs.

In exemplary embodiments, one or more additional drugs comprise one ormore glucose lowering drugs. In various embodiments, one or moreadditional drugs comprise one or more glucose lowering drugs comprisinga drug or combination of drugs selected from a glitazone, a GLP-1analog, a DPPIV inhibitor, metformin, a glinide, a sulfonylurea, anα-glucosidase inhibitor and an insulin. In various embodiments, theinsulin is selected from a regular human insulin, an intermediate-actingregular human insulin, a Zn-retarded insulin, a short-acting insulinanalog and a long-acting insulin analog. In exemplary embodiments, oneor more additional drugs comprise one or more glucose lowering drugsexcluding a drug or combination of drugs selected from a sulfonylurea, aglinide and a regular human insulin.

In exemplary embodiments, one or more glucose lowering drugs comprise adrug or combination of drugs selected from a DDPIV inhibitor, metforminand an α-glucosidase inhibitor. In exemplary embodiments, one or moreglucose lowering drugs comprise a drug or combination of drugs selectedfrom metformin and an α-glucosidase inhibitor. In exemplary embodiments,a glucose lowering drug is an α-glucosidase inhibitor. In exemplaryembodiments, one or more glucose lowering drugs comprise a drug orcombination of drugs selected from a GLP-1 analog, a glinide, asulfonylurea and an α-glucosidase inhibitor.

In various embodiments, one or more drug is combined with one or moretreatment regimens such as diet, exercise and so on.

The concept of a pathophysiological oriented therapy of type 2 diabeteswith effective insulin resistance treatment shows beneficialanti-inflammatory and anti-thrombotic effects and should clearly bepreferred to measures of “glucose cosmetics.” Marker panels describinginsulin resistance, 3-cell dysfunction, adipogenesis and atherosclerosismay be more predictive and meaningful than HbAlc.

A combination of β-cell protective agents (for example, an insulin, aDPPIV inhibitor, a GLP-1 analog) might turn type 2 diabetes into anon-progressive disease. Table 5 shows the effect of a number ofcompounds on levels of biomarkers adiponectin, hsCRP and intactproinsulin. An up arrow indicates an increase in biomarker levels, adown arrow indicates a decrease and “O” indicates substantially nochange. Thus, in one embodiment, a subject is administered asulfonylurea, a glinide, metformin, a DPPIV-inhibitor, a GLP-1 analog, aglitazone or an insulin or combinations thereof for treatment ofcardiodiabetes.

TABLE 6 hsCRP intact proinsulin adiponectin sulfonylurea ↑ ↑ ↓ glinides↑ ↑ ↓ metformin O ↓ ↑ DPPIV-inhibitor O ↓ O GLP-1 analog O ↓ O TZD ↓ ↓ ↑insulin O ↓ O

Decision Matrices

The therapy chosen by a practitioner can depend on the concentrations ofbiomarkers determined in a sample. In various exemplary embodiments, thetherapy depends on which category from a range of categories particularto each biomarker the measured concentration of each biomarker falls in.In various exemplary embodiments, the therapy depends on the combinationof risk levels for different symptoms or diseases that are indicated bya biomarker panel.

With respect to concentration measurements of a biomarker, the term“category” refers to a subset of a partition of the possibleconcentrations that a biomarker may have. Each category may beassociated with a label or classification chosen by the practitioner.The labels may be refer to, for example, the risk level of an individualfor having or being subject to a disease state. The categories andlabels may be derived from the current literature or according to thefindings of the practitioner. For example, it is known in the art thatan individual with a serum concentration of adiponectin that is greaterthan 10 mg/L has a low risk for arteriosclerosis and insulin resistance,while an individual with a serum concentration of 7-10 mg/L has a mediumrisk for these disorders. An adiponectin concentration of greater than10 mg/L thus can be labeled a “low risk” concentration and that of 7-10mg/L a “medium risk” or “moderate risk” concentration. Table 2A showsthat adiponectin concentrations can be divided into three categories forthe purposes of the methods described herein. The number of categoriesand the boundaries dividing them may vary. For example, Table 2B showsan alternative categorization for adiponectin. The number of categoriesand the boundaries dividing them for any biomarker are not limited tothose specifically disclosed herein and can be found in the art.

Each biomarker of a biomarker panel can thus be associated with adiscrete set of categories, for example, risk categories. Combining onecategory from each biomarker forms a “decision point.” In variousexemplary embodiments, the complete set of decision points comprises allpossible n-tuples of categories, wherein n is the number of biomarkersin the biomarker panel. This complete set will have m₁×m₂× . . . m_(n)possible decision points, wherein m_(i) is the number of categories forbiomarker i.

Every decision point can be associated with a condition or a diseasestate, which is not necessarily unique. That is, one or more decisionpoints can be associated with the same disease state. The association ofevery possible decision point with a condition or disease state can bereferred to as a “disease classification matrix” or a “diseaseclassification tree.” Thus, by correlating a measurement of a biomarkerpanel with a decision point, the practitioner can classify the conditionor disease state of a patient.

Every decision point can also be associated with a particular therapy,which is not necessarily unique. That is, one or more decision pointscan be associated with the same therapy. The association of everypossible decision point with one or more therapies can be referred to asa “therapy decision matrix” or “therapy decision tree.”

Each decision point can be associated with more than one type ofinformation. For example, both disease state and therapy can beindicated by a decision point.

In various exemplary embodiments, the biomarker panel consists ofadiponectin, hsCRP and intact proinsulin. In various exemplaryembodiments, possible concentrations of adiponectin in a sample aredivided into 3 risk categories. In various exemplary embodiments,possible concentrations of hsCRP in a sample are divided into 3 riskcategories. In various exemplary embodiments, possible concentrations ofintact proinsulin in a sample are divided into 2 risk categories. Invarious exemplary embodiments, the therapy decision matrix consists of18 decision points, each associated with a therapy that may or may notbe unique across the set of all therapies.

In various exemplary embodiments, the risk categories for adiponectinare provided by Table 2A. In various exemplary embodiments, the riskcategories for hsCRP are provided by Table 3. In various exemplaryembodiments, the risk categories for intact proinsulin are provided byTable 4.

In various exemplary embodiments, if the risk level associated with eachof the concentrations of adiponectin, hsCRP and intact proinsulin in asample respectively is selected from (a) high, high and high; (b) high,medium and high; (c) medium, high and high; (d) low, high and high; (e)low, medium and high; and (f) low, low and high, then the subject isadministered a glitazone and a drug or combination of drugs selectedfrom a GLP-1 analog and an insulin. In various exemplary embodiments,the subject is not administered a drug or combination of drugs selectedfrom a sulfonylurea and a glinide. In various exemplary embodiments, thesubject is optionally administered one or more glucose lowering drugs.In various exemplary embodiments, the subject is optionally administeredone or more glucose lowering drugs comprising a drug or combination ofdrugs selected from a DDPIV inhibitor, metformin and an α-glucosidaseinhibitor.

In various exemplary embodiments, if the risk level associated with eachof the concentrations of adiponectin, hsCRP and intact proinsulin in asample respectively is selected from (a) high, low and high; (b) medium,medium and high; and (c) medium, low and high, then the subject isadministered a glitazone and a drug or combination of drugs selectedfrom a GLP-1 analog, an insulin and a DPPIV inhibitor. In variousexemplary embodiments, the subject is not administered a drug orcombination of drugs selected from a sulfonylurea and a glinide. Invarious exemplary embodiments, the subject is optionally administeredone or more glucose lowering drugs. In various exemplary embodiments,the subject is optionally administered one or more glucose loweringdrugs comprising a drug or combination of drugs selected from metforminand an α-glucosidase inhibitor.

In various exemplary embodiments, if the risk level associated with eachof the concentrations of adiponectin, hsCRP and intact proinsulin in asample respectively is selected from (a) high, high and low; (b) medium,high and low; (c) low, high and low; (d) low, medium and low; and (e)low, low and low, then the subject is administered metformin and a drugor combination of drugs selected from a glitazone, a GLP-1 analog, aDPPIV inhibitor and an insulin. In various exemplary embodiments, thesubject is not administered a drug or combination of drugs selected froma sulfonylurea and a glinide. In various exemplary embodiments, thesubject is optionally administered one or more glucose lowering drugs.In various exemplary embodiments, the subject is optionally administereda glucose lowering drug comprising an α-glucosidase inhibitor.

In various exemplary embodiments, when the concentration of adiponectinin a sample from a subject is if the risk level associated with each ofthe concentrations of adiponectin, hsCRP and intact proinsulin in asample respectively is selected from (a) high, medium and low; (b) high,low and low; (c) medium, medium and low; and (d) medium, low and low,then the therapy comprises administering metformin and a drug orcombination of drugs selected from a glitazone, a DPPIV inhibitor and aninsulin. In various exemplary embodiments, the subject is optionallyadministered one or more glucose lowering drugs. In various exemplaryembodiments, the subject is optionally administered one or more glucoselowering drugs comprising a drug or combination of drugs selected from aGLP-1 analog, a glinide, a sulfonylurea and an α-glucosidase inhibitor.

In one aspect, the invention provides drugs for the treatment of adisease. In exemplary embodiments, these drugs are administered to asubject for which concentrations of each biomarker of a biomarker panelin a sample from the subject correspond to a decision point providedherein.

In various embodiments, the drugs comprise a glitazone and a drug orcombination of drugs selected from a GLP-1 analog and an insulin fortreating a disease in a subject for which concentrations of a biomarkerpanel in a sample from the subject are measured. In various exemplaryembodiments, the biomarker panel consists of adiponectin, hsCRP andintact proinsulin, and the risk level associated with each of theconcentrations of adiponectin, hsCRP and intact proinsulin respectivelyis selected from (a) high, high and high; (b) high, medium and high; (c)medium, high and high; (d) low, high and high; (e) low, medium and high;and (f) low, low and high. In various exemplary embodiments, the drugsdo not include a drug or combination of drugs selected from asulfonylurea and a glinide. In various exemplary embodiments, the drugsoptionally further comprise one or more additional drugs selected fromone or more glucose lowering drugs. In various exemplary embodiments,one or more glucose lowering drugs comprise a drug or combination ofdrugs selected from a DPPIV inhibitor, metformin and an α-glucosidaseinhibitor.

In various embodiments, the drugs comprise a glitazone and a drug orcombination of drugs selected from a GLP-1 analog, an insulin and aDPPIV inhibitor for treating a disease in a subject for whichconcentrations of a biomarker panel in a sample from the subject aremeasured. In various exemplary embodiments, the biomarker panel consistsof adiponectin, hsCRP and intact proinsulin, and the risk levelassociated with each of the concentrations of adiponectin, hsCRP andintact proinsulin respectively is selected from (a) high, low and high;(b) medium, medium and high; and (c) medium, low and high. In variousexemplary embodiments, the drugs do not include a drug or combination ofdrugs selected from a sulfonylurea and a glinide. In various exemplaryembodiments, the drugs optionally further comprise one or moreadditional drugs selected from one or more glucose lowering drugs. Invarious exemplary embodiments, one or more glucose lowering drugscomprise a drug or combination of drugs selected from metformin and anα-glucosidase inhibitor.

In various embodiments, the drugs comprise metformin and a drug orcombination of drugs selected from a glitazone, a GLP-1 analog, a DPPIVinhibitor and an insulin for treating a disease in a subject for whichconcentrations of a biomarker panel in a sample from the subject aremeasured. In various exemplary embodiments, the biomarker panel consistsof adiponectin, hsCRP and intact proinsulin, and the risk levelassociated with each of the concentrations of adiponectin, hsCRP andintact proinsulin respectively is selected from (a) high, high and low;(b) medium, high and low; (c) low, high and low; (d) low, medium andlow; and (e) low, low and low. In various exemplary embodiments, thedrugs do not include a drug or combination of drugs selected from asulfonylurea and a glinide. In various exemplary embodiments, the drugsoptionally further comprise one or more additional drugs selected fromone or more glucose lowering drugs. In various exemplary embodiments,one or more glucose lowering drugs comprise one or more glucose loweringdrugs comprising a drug or combination of drugs selected from metforminand an α-glucosidase inhibitor.

In various embodiments, the drugs comprise metformin and a drug orcombination of drugs selected from a glitazone, a DPPIV inhibitor and aninsulin for treating a disease in a subject for which concentrations ofa biomarker panel in a sample from the subject are measured. In variousexemplary embodiments, the biomarker panel consists of adiponectin,hsCRP and intact proinsulin, and the risk level associated with each ofthe concentrations of adiponectin, hsCRP and intact proinsulinrespectively is selected from (a) high, medium and low; (b) high, lowand low; (c) medium, medium and low; and (d) medium, low and low. Invarious exemplary embodiments, the drugs optionally further comprise oneor more additional drugs selected from one or more glucose loweringdrugs. In various exemplary embodiments, one or more glucose loweringdrugs comprise one or more glucose lowering drugs comprising a drug orcombination of drugs selected from a GLP-1 analog, a glinide, asulfonylurea and an α-glucosidase inhibitor.

The articles “a,” “an” and “the” as used herein do not exclude a pluralnumber of the referent, unless context clearly dictates otherwise. Theconjunction “or” is not mutually exclusive, unless context clearlydictates otherwise. The term “include” is used to refer to non-limitingexamples.

All references, publications, patent applications, issued patents,accession records and databases cited herein, including in anyappendices, are incorporated by reference in their entirety for allpurposes.

We claim:
 1. A composition comprising a solid support comprising: (a) acapture binding ligand selective for adiponectin, (b) a capture bindingligand selective for hsCRP, and (c) a capture binding ligand selectivefor intact proinsulin.
 2. The composition of claim 1 wherein one of thecapture binding ligands comprises an antibody.
 3. The composition ofclaim 1 wherein the composition further comprises: (a) a soluble captureligand selective for adiponectin, (b) a soluble capture ligand selectivefor hsCRP, and (c) a soluble capture ligand selective for intactproinsulin.
 4. The composition of claim 3 wherein each of the solublecapture ligands comprises a detectable marker.
 5. The composition ofclaim 4 wherein a detectable marker is a fluorophore.
 6. The compositionof claim 4 wherein a detectable marker is a conjugated enzyme.
 7. Thecomposition of claim 6 Wherein the conjugated enzyme is horseradishperoxidase.
 8. The composition of claim 1 further comprising a detector.9. A method of treating cardiodiabetes in a subject comprising (a)measuring the concentration of a biomarker panel in a sample from thesubject, the biomarker panel consisting of adiponectin, hsCRP and intactproinsulin; and (b) effecting a therapy with respect to the subject. 10.The method of claim 9 wherein if the risk level associated with each ofthe concentrations of adiponectin, hsCRP and intact proinsulinrespectively is selected from (a) high, high and high; (b) high, mediumand high; (c) medium, high and high; (d) low, high and high; (e) low,medium and high; and (0 low, low and high, then the subject isadministered a glitazone and a drug or combination of drugs selectedfrom a GLP-1 analog and an insulin.
 11. The method of claim 9 wherein ifthe risk level associated with each of the concentrations ofadiponectin, hsCRP and intact proinsulin respectively is selected from.(a) high, low and high; (b) medium, medium and high; and (c) medium, lowand high, then the subject is administered a glitazone and a drug orcombination of drugs selected from a GLP-1 analog, an insulin and aDPPIV inhibitor.
 12. The method of claim 9 if the risk level associatedwith each of the concentrations of adiponectin, hsCRP and intactproinsulin respectively is selected from (a) high, high and low; (b)medium, high and low; (c) low, high and low; (d) low, medium and low;and (e) low, low and low, then the subject is administered metformin anda drug or combination of drugs selected from a glitazone, a GLP-1analog, a DPPIV inhibitor and an insulin,
 13. The method of claim 9wherein it' the risk level associated with each of the concentrations ofadiponectin, hsCRP and intact proinsulin respectively is selected from(a) high, medium and low; (b) high, low and low; (c) medium, medium andlow; and (d) medium, low and low, then the therapy comprisesadministering metformin and a drug or combination of drugs selected froma glitazone, a DPPIV inhibitor and an insulin.
 14. The method of any ofclaims 9-12 wherein the subject is not administered a drug orcombination of drugs selected from a sulfonylurea and a glinide.
 15. Themethod of any of claims 9-14 wherein the subject is administered one ormore additional drugs comprising one or more glucose lowering drugs. 16.The method of claim 9 wherein the sample comprises blood.
 17. The methodof claim 9 claim further comprising taking a measurement of at least oneadditional biomarker.
 18. The method of claim 17 wherein the additionalbiomarker is selected from the group consisting of leptin, mRNAx, NFκB,m IL-6, MMP-9, TNFα, NFκB, eNOS, PPARγ, MCP-1, PAI-1, ICAM/VCAM,E-selectin, P-selectin, von Willebrand factor, sCD40L, insulin, glucose,HbAlc, free fatty acids, triglycerides, VLDL, small dense LDL, oxidizedLDL, resistin, HDL, NO, IκB-α, p105, Re1A, TNFα, MIF, inflammatorycytokines and molecules involved in signaling pathways.
 19. The methodof claim 18 wherein the additional biomarker is MMP-9.
 20. The method ofclaim 19 wherein the concentration of intact proinsulin, hsCRP andadiponectin in the sample is measured and each is a high riskconcentration.
 21. The method of claim 18 wherein the additionalbiomarker is leptin.
 22. A method of treating cardiodiabetes in asubject comprising: (a) contacting a sample from the subject with thecomposition of claim 1: (b) measuring the concentration of a biomarkerpanel in the sample from the subject, the biomarker panel consisting ofadiponectin, hsCRP and intact proinsulin: and (c) effecting a therapywith respect to the subject.